whacky ppt/quant results - (Nov/02/2005 )
I'm using a standard T4 dna ligase protocol to make a molecular padlock (combining a 42mer target with a 95mer probe, which yields a circular padlock having a 42 double-base paired region and a 53 single-base region), followed by exonuclease treatment to remove unligated probe and target. Gel analysis (and subsequent experiments) shows that padlock construction is working perfectly. It's the UV quantitation that is nuts! I'm etoh ppt the ligation mix using sodium acetate, wash with 70% etoh, get a nice pellet that resuspends in H2O quickly and easily. However, the A260 indicates that I have several of orders of magnitude MORE nucleic acid than I started with. I suspect (a) ATP in ligation buffer is precipitating and giving false readings, or ( circular double/single stranded nature of padlock makes A260 readings invalid. DO dNTPs precipitate in an etoh ppt? Does anyone have any insight on what might be happening here or things to look at? Thanks.
first, you can't have a 42dble pair region and a 53 single strand region because you need nucleotides that forms the hairpin structure... or i'm not getting all stuff
what are the 260:230 (aromatic acids) and 260:280 (proteins) ratio values?
This is not a hairpin or a dumbbell. 21 bases on the 5' end of the probe are complimentary to the 3'half of the target; 21 bases on the 3' end of the probe are complimentary to the 5' half of the target. The distal 5' and 3' base of the probe meet in the middle of the target and can be ligated. This makes the target region double stranded and the loop portion (i.e., from base 22-74 of the probe sequence) single stranded. Simple in approach, difficult to explain with words (but easy with pictures).
It's not the padlock construction that concerns me; it's the question of whether the atp from the ligation buffer precipitates with the padlock and jerks up the A260 or if it's the circular nature of the padlock itself that screws up the reading. A260= .1074, A280= .0136, 1:100 diln in TRIS pH7.6